Methods for treating subjects infected with a herpes virus

ABSTRACT

The present invention provides a method of inhibiting the formation of infectious herpes virus particles, particularly infectious herpes simplex virus (HSV) particles, in a host cell. The method involves administering an effective amount of a hydroxylated tolan, particularly a polyhydroxylated tolan, to a herpes virus infected host cell. The present invention also provides a method of treating a herpes virus infection, particularly an HSV infection. The method comprises administering a topical composition comprising a therapeutically effective amount of a hydroxylated tolan to a herpes virus-infected site. The present invention also relates to a topical composition for treating a herpes virus infection selected from the group consisting of an HSV infection, a cytomegalovirus infection, and a varicella zoster virus infection. The present invention also provides a method of treating a subject infected with Neisseria gonorrhea.

This application claims priority to U.S. Provisional Application No.60/225,609, filed Aug. 15, 2000.

BACKGROUND

The present invention relates to compositions which inhibit replicationof herpes virus and the bacterium Neisseria gonorrheae, and methods ofusing such compositions to treat subjects infected with thesemicroorganisms.

Human herpes viruses can infect host cells in virtually any organ of thehuman body. Replication of a herpes virus within an infected host cellleads to lysis of the infected cell and the release of large numbers ofinfectious virus. The infectious particles released from the lysed cellcan infect and destroy other cells at or near the site of the initialinfection. These infectious particles can also be transmitted to anon-infected individual. Human herpes viruses can also enter and remainlatent, i.e., in the non-replicative state, in other cells of theafflicted individual for life. This life-long infection serves as areservoir of infectious virus for recurrent infections in the afflictedindividual and as a source of infection for an unwitting contact.

At least four of the human herpes viruses, including herpes simplexvirus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2),cytomegalovirus (CMV), and varicella zoster virus (VZV) are known toinfect and cause lesions in the eye of certain infected individuals.Together, these four viruses are the leading cause of infectiousblindness in the developed world.

HSV-1 primarily infects the oral cavity, while HSV-2 primarily infectsgenital sites. However, any area of the body, including the eye, skinand brain, can be infected with either type of HSV. Generally, HSV istransmitted to a non-infected individual by direct contact with theinfected site of the infected individual.

The initial symptoms of a primary or recurrent HSV infection includetingling, pain, and/or parasthesia at the site of infection. This isfollowed by formation of a lesion at the infected site, i.e., in theoral cavity, eye, skin, or reproductive tract. Healing typically occursin approximately ten to fourteen days.

The immune reaction that occurs in response to an HSV infection preventsdissemination of the virus throughout the body of the immmunocompetentindividual. Such immune reaction, however, does not eliminate allinfectious HSV particles from the body of the afflicted individual. Thevirus particles that are not killed by the immune response move alongthe nerve path to the ganglia of the infected individual where theyremain in a state of latency. In response to a variety of stimuliincluding stress, environmental factors, other medications, foodadditives or food substances, the infectious virus particles may leavethe ganglia and cause a recurrent infection at or near the original siteof infection. In those HSV-infected individuals who are immunosuppressedor who lack a well-developed immune system, such as neonates,dissemination of the virus particles from the infected site can alsooccur and lead to life-threatening complications, includingencephalitis.

VZV, which is transmitted by the respiratory route, is the cause ofchickenpox, a disease which is characterized by a maculopapular rash onthe skin of the infected individual. As the clinical infection resolves,the virus enters a state of latency in the ganglia, only to reoccur insome individuals as herpes zoster or “shingles”. The reoccurring skinlesions remain closely associated with the dermatome, causing intensepain and itching in the afflicted individual.

CMV is more ubiquitous and may be transmitted in bodily fluids. Theexact site of latency of CMV has not been precisely identified, but isthought to be leukocytes of the infected host. Although CMV does notcause vesicular lesions, it does cause a rash.

There are no known cures for infections with human herpes viruses, i.e.,methods of eliminating the virus from the body of the infectedindividual. In addition, there are very few methods for blocking theformation of infectious herpes virus particles and thereby reducing thefrequency, severity, or duration of a herpes virus-induced infection andthe likelihood of recurrence of infection in the latently-infectedindividual. Thus, it is desirable to have additional methods forinhibiting the formation of infectious herpes virus particles. Suchmethod is useful for limiting the severity of a herpes virus infectionwithin an infected individual and the likelihood of transmission of theherpes virus infection from the infected individual to a non-infectedindividual.

Neisseria gonorrhea is a gram negative bacterium that is pathogenic inhumans. The bacterium is spread from person to person by contact withinfected secretions, most often by sexual contact. Once the pathogen isdeposited on a mucosal surface, a complex series of molecularinteractions occur that result in invasion of mucosal columnar cells.The spectrum of diseases ranges from local infections of the urethral,cervical, rectal and oropharyngeal membranes to invasion of the pelvisor epididymis, to invasion of the blood stream, with or withoutdissemination to distant organs such as heart valves, joints, andpericardium. The pathogen may also infect the conjunctiva. Gonococcalconjunctivitis is most often contracted by neonates passing through aninfected birth canal, although adults can also be infected.

The quest for a gonococcal vaccine has been ongoing for many years withvirtually no success. Accordingly, the primary treatment involvespreexposure or postexposure antibiotic prophylaxis. In addition toantibiotic eyedrops, silver nitrate has also been used to treat neonatalgonococcal conjunctivitis. Unfortunately, the bacterium has developedresistance to some of the most common antibiotics, such as penicillin.Accordingly, additional compositions for reducing growth of thispathogen are desirable.

SUMMARY OF THE INVENTION

The present invention provides a new method of inhibiting the formationof infectious herpes virus particles, particularly infectious HSVparticles, in a host cell. The method involves administering ahydroxylated tolan, particularly dihydroxytolan or trihydroxytolan, to aherpes virus infected host cell. The hydroxylated tolan is administeredto the host cell in an amount sufficient to inhibit replication of thevirus in the virus-infected host cell. Such method is useful forreducing the cytopathic effect of a herpes virus infection. Such methodis also useful for preventing the spread of the herpes virus from avirus-infected host cell to a non-infected host cell. Such method isalso useful for establishing a model system for studying the molecularevents that occur during replication of herpes virus and for studyingthe factors that trigger replication of a latent herpes virus,particularly replication of latent HSV.

The present invention also provides a method of treating a subjecthaving or suspected of having a herpes virus infection, particularly anHSV infection. The method comprises administering a topical compositioncomprising a therapeutically effective amount of a hydroxylated tolan,particularly a di-hydroxylated or tri-hydroxylated tolan, to a herpesvirus-infected site. The present invention also relates to a topicalcomposition for treating a herpes virus infection selected from thegroup consisting of an HSV infection, a CMV infection, and a VZVinfection.

The present invention also provides a method of inhibiting replicationof the gram negative bacterium Neisseria gonorrhea. Such method involvescontacting the bacterium with a composition containing a hydroxylatedtolan, preferably a di-hydroxylated or tri-hydroxylated tolan. In vivo,such method can be used to treat an individual who has come in contactwith, (e.g., a carrier), or an individual who is expected to come intocontact with the bacterium. In vivo, such method comprises administeringa composition comprising a therapeutically effective amount of ahydroxylated tolan, particularly a tri-hydroxylated tolan to the subject

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of 4,4′ dihydroxytolan (“Tolan-5”),3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (“Tolan-11).

FIG. 2 is a synthetic scheme for making hydroxylated tolans.

FIG. 3 is a synthetic scheme for making hydroxylatedtrifluoromethyltolans.

FIG. 4 is a graph showing the extent of HSV-1 replication invirus-infected cells treated with different concentrations of adihydroxylated tolan.

FIG. 5 is a graph showing the extent of HSV-1 replication invirus-infected cells treated with 4,4′ dihydroxytolan at different timesfollowing infection.

FIG. 6 is a graph depicting the reversibility of the inhibitory effectof 4,4′-dihydroxytolan on HSV-1 replication in virus-infected cells.

FIG. 7 is a graph depicting the effect of treatment with4,4′-dihydroxytolan on the accumulation of ICP-4 in HSV-1 infectedcells.

FIG. 8 is a graph depicting the effect of treatment with4,4′-dihydroxytolan on the accumulation of ICP-27 in HSV-1 infectedcells.

FIGS. 9A, 9B, and 9C are graphs showing the inhibitory effects of 4,4′dihydroxytolan (“Tolan-5”), 3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (Tolan-11), respectively, onreplication of HSV-1 in infected cells.

FIG. 10 is a graph showing the effect of a 5% Tolan-10 solution on thedevelopment of herpetic lesions in animals infected with HSV-1.

FIG. 11 is a graph showing the effect of a 10% Tolan-10 solution on thedevelopment of herpetic lesions in animals infected with HSV-1.

FIG. 12 is a graph showing the percentage of control animals andtolan-treated animals that survive an infection with HSV-1.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method of inhibitingformation of infectious herpes virus particles, particularly infectiousHSV particles, in a host cell. The method comprises administering ahydroxylated tolan to the host cell. The hydoxylated tolan isadministered in an amount sufficient to or effective to inhibitreplication of the herpes virus within the infected cell. Preferably,the hydroxylated tolan, is administered to the host cell either prior toinfection of the host cell with the virus or within six hours afterinfection of the host cell with the virus.

Preferably, the hydroxylated tolan is administered to the host cell bycontacting the host cell with or exposing the host cell to a compositioncomprising the hydroxylated tolan. For example, in vitro, the methodcomprises adding a hydroxylated tolan to the culture medium of herpesvirus-infected host cells. In the case of cultured cells, thehydroxylated tolan preferably is added to the medium before the hostcells are infected with the virus or within six hours after the hostcells are infected with the virus. In the case of ganglia, which servesas an organ culture model system for studying latency of herpes viruses,particularly for studying latency of HSV, the hydroxylated tolan isadded to the medium after the ganglia are excised from thelatently-infected host.

It has been determined that treatment of cultured cells in accordancewith the present method is non-toxic to cells and blocks replication ofHSV at some early stage in the replicative cycle of this human herpesvirus. It has also been determined that the effect of a polyhydroxylatedtolan on HSV replication is reversible. Typical of the herpes viruses,HSV replication occurs in phases, with each phase being dependent on thesuccessful completion of the prior phase. The “immediate early phase”occurs at 1-3 hours after infection and is associated with regulatoryand synthetic events. The “early phase” occurs 3-6 hours after infectionand is also associated with regulatory and synthetic events,particularly the synthesis of virus DNA. The “late phase” occurs 6-10hours after infection and is associated with final synthetic events andassembly of viral components into infections virions. Accordingly, sinceall herpes viruses have in common a replicative scheme that progressesthrough similar and distinct phases, such method is useful forestablishing model systems for studying the molecular events that occurduring replication of all herpes viruses. For example, mammalian cellcultures incubated in the presence and absence of a hydroxylated tolanmay be used to identify cellular factors that are involved in regulatingherpes virus synthetic events. Such cell cultures may also be employedto characterize the role of HSV gene products in the replication ofinfectious virus, particularly those proteins and factors whose functionare currently unknown.

Such method is also useful for establishing a model system for studyinglatency of herpes viruses, particularly latency of the herpes virusesthat remain latent in the ganglia, such as for example HSV and VZV. Suchmodel system is useful for characterizing the extracellular factors suchas for example hormones and cytokines, as well as the intracellularfactors and molecular events that trigger replication of latent herpesviruses.

Methods and Compositions for Treating a Subject With a HerpesvirusInfection

In another aspect, the present invention provides methods for treating asubject with a herpesvirus infection. The method comprises administeringa pharmaceutical composition, preferably a topical composition,comprising a therapeutically effective amount of a hydroxylated tolan,preferably a dihydroxylated tolan or trihydroxylated tolan, to the siteof the infection. As used herein “site of the infection” means apreviously uninfected site which is expected to come into contact with aherpes virus-infected site or the site of a current or prior herpesvirus-induced lesion. Such method is particularly useful for treatinglocal herpes virus infections, such as for example, HSV-induced skinlesions, HSV-induced eye infections, HSV-induced lesions of thereproductive tract, CMV-induced eye lesions, and VZV-induced eyelesions. In such cases, it is preferred that the hydroxylated tolan beapplied directly to the infected site. It is preferred that thehydroxylated tolan be administered to the herpesvirus-infected site inthe form of an aqueous solution or in the form of a salve. For eyeinfections, it is preferred that an aqueous solution of the hydroxylatedtolan, be administered as an eye drop. For herpesvirus skin lesions,such as for example, HSV-induced skin lesions, or HSV-induced lesions ofthe reproductive tract, it is preferred that the composition be appliedtopically.

Method of Inhibiting Growth of Neisseria gonorrheae

In another aspect, the present invention provides a method of inhibitingthe growth of Neisseria gonnorhea. The method comprises administering ahydroxylated tolan, preferably a dihydroxlated or trihydroxylated tolanto a surface which has come in contact with or could come in contactwith the organism. In vivo, the method, which comprises administeringthe hydroxylated tolan to a mucous membrane of a human subject, may beused to prevent or reduce the symptoms of gonococcal disease in thehuman subject. The hydroxylated tolan may be incorporated into apharmaceutical composition which is applied to the mucous membrane of acarrier of the bacterium or a person who could come into contact withthe carrier.

Administration of the pharmaceutical composition to an uninfectedsubject is via local administration to a site which has been or may becontacted with the pathogenic organism. It is preferred that thepharmaceutical composition be applied prior to exposure to the targetedpathogen or preferably within 1-24 hours, more preferably within 1-12hours after exposure of the uninfected subject to the pathogenicorganism. Administration of the pharmaceutical composition to a carrierof Neisseria gonorrhea is via local administration to the genitalia,rectum, or oropharynx.

Hydroxylated Tolans

The structural skeleton of the compound employed in the presentinvention, i.e., the hydroxylated tolan, comprises two aromatic ringsjoined by an acetylene bridge. Preferably, the hydroxylated tolan is apolyhydroxylated tolan, more preferably a dihydroxytolan, or atrihydroxytolan, most preferably a trihydroxytolan. A general scheme forpreparing polyhydroxylated tolans is shown in FIG. 2. A general schemefor preparing a hydroxylated trifluormethyltolan is shown in FIG. 3.

Topical Composition

The pharmaceutical composition comprises a therapeutically effectiveamount of a hydroxylated tolan, preferably a polyhydroxylated tolan,more preferably a dihydroxylated or trihyroxylated tolan, and apharmaceutically acceptable carrier, preferably a topical carrier.Preferably, the composition comprises a relatively inert topicalcarrier. Many such carriers are routinely used and can be identified byreference to pharmaceutical texts. Examples include polyethyleneglycols, polypropylene copolymers, and some water soluble gels. Such acomposition, referred to hereinafter as the “topical composition”, mayalso contain diluents, fillers, salts, buffers, stabilizers,solubilizers, and other pharmaceutically acceptable materials well knownin the art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of the antiviralor antibacterial activity of the hydroxylated tolan.

In practicing the present method of treatment or use, a pharmaceuticalcomposition comprising a therapeutically effective amount of thehydroxylated tolan is applied to the site of infection in the hostsubject before or after the host subject is exposed to the virus orbacterium. Such composition is particularly effective in treatinginfections of the eye, oral cavity and vagina as well as border areas ofthe lips and rectum. In the case of oral administration, dentrifices,mouthwashes, tooth paste or gels, or mouth sprays are used. Vaginal orrectal administration may be by the usual carriers such as douches,foams, creams, ointments, jellies, and suppositories, the longer lastingforms being preferred. Ocular administration is preferably by ophthalmicointments or solutions. Lip treatment is, preferably, in the form of agel.

The topical composition may further contain other agents which eitherenhance the activity of the hydroxylated tolan or complement itsactivity or use in treating the viral disease or bacterial disease. Suchadditional factors and/or agents may be included in the pharmaceuticalcomposition to produce a synergistic effect with the hydroxylated tolan,or to minimize side effects. The topical composition may also contain anagent which enhances uptake of the hydroxylated tolan.

Preferably the topical composition comprises a solvent for thehydroxylated tolan, such as, for example, an alcohol. A liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oil, soybean oil, or sesame oil, corn oil, or syntheticoils may be added. The liquid form of the pharmaceutical composition mayfurther contain a physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. The preparation of such topicalcomposition having suitable pH, isotonicity, and stability, is withinthe skill in the art.

The topical composition of the invention may be in the form of aliposome in which the hydroxylated tolan is combined with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art.

Dosage

The hydroxylated tolan is administered to the site of infection in thehost subject in a therapeutically effective amount. As used herein, theterm “therapeutically effective amount” means the total amount of thehydroxylated tolan that is sufficient to show a meaningful benefit,i.e., treatment, healing, prevention, amelioration, or reduction in thesymptoms of the herpesvirus or gonococcal infection or an increase inrate of healing, amelioration or reduction in the symptoms of suchinfection.

By “treating” is meant curing or ameliorating a herpesvirus orgonococcal infection or tempering the severity of the infection. Bypreventing is meant blocking the formation of a primary lesion orrecurrence of a lesion at the infected site. The dosages of thehydroxylated tolan, which can treat or prevent an HSV, VZV, CMVinfection, or gonococcal infection, can be determined in view of thisdisclosure by one of ordinary skill in the art by running routine trialswith appropriate controls. Comparison of the appropriate treatmentgroups to the controls will indicate whether a particular dosage iseffective in preventing or treating the infection at the levels used ina controlled challenge.

It is contemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.1 μgto about 100 mg/ml. Although a single application of the topicalcomposition may be sufficient to ameliorate the pathological effects ofthe herpesvirus or Nisseria gonorrhea, it is expected that multipledoses will be preferred.

Delivery

Administration of the pharmaceutical composition is via localadministration to the infected site. In those individuals who haveexperienced a primary lesion, it is preferred that the topicalcomposition be applied at the prodromal stage of infection, i.e., duringearly symptoms of pain, tingling, parasthesia. Preferably, thecomposition is applied to the site of infection periodically, morepreferably every three hours. The duration of therapy using thepharmaceutical composition of the present invention will vary, dependingon the severity of the disease being treated and the condition andresponse of each individual patient. Ultimately the attending physicianwill decide on the appropriate duration of using the pharmaceuticalcomposition of the present invention.

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims which are appended hereto. Allreferences cited herein are specifically incorporated in their entiretyherein.

EXAMPLE 1 Synthesis of Poly-Hydroxylated Tolans

A. Synthesis of 3,5-dimethoxyiodobenzene from 3,5-dimethoxyaniline

In a 500 ml 3-necked, round-bottomed flask equipped with a thermometer,a mechanical stirrer and an addition funnel was placed HCl (12 M, 100ml, 1.2 mol) and crushed ice (100 g). The flask was immersed in a dryice-Me₂CO cooling bath, and 3,5-dimethoxyaniline (15.3 g, 100 mmol) wasadded with stirring. To this cold mixture NaNO₂ (8.4 g, 120 mmol) in 40ml H₂O was added dropwise at such a rate to maintain the temperature ofthe reaction mixture between −10˜−5° C. throughout the addition. Thereaction mixture was stirred for 1 hour at 0˜5° C. The red dark solutionof the diazonium salt was added to a well-stirred solution of KI (83 g,500 mmol) in 200 ml H₂O at room temperature. The mixture was stirred for2 hours, then allowed to stand overnight. The resulting solution wasextracted with ether (200 ml×4). The pooled organic extracts were washedwith brine (200 ml×2), and an aqueous saturated Na₂S₂O₃ solution (200ml×2), dried over MgSO4, filtered and concentrated to a small volume.Silica gel was added, and the mixture evaporated to dryness. Thispreloaded silica gel was placed on a pad of silica gel and eluted withpetroleum to give 17.5 g (66%) of a colorless solid, 3,5-dimethoxyiodobenzene. ¹HNMR (CDCl₃, 300 Mz): δ ppm: 6.85 (2H, d, J=2.3, Ar—H), 6.40(1H, t, J=2.3 Ar—H), 3.76 (s, 6H, 2CH₃O).

B. Synthesis of Arylethynyltrimethylsilanes from Ethylnyltrimethylsilaneand Aryl Iodides

General Procedure

To a solution of aryl methoxy substituted aryl iodide (40 mmol) inisopropylamine (250 ml) were added Pd(PPh₃)₂Cl₂(0.4 mmol) and CuI (0.8mmol), then trimethylsilylacetylene (44 mmol). The reaction mixture wasstirred at ambient temperature for 2-4 hours under a slow stream ofnitrogen. The reaction mixture was filtered and the residues were washedwith ethyl acetate, and the solvent evaporated from the combinedfiltrates. The crude product was purified by column chromatography onsilica gel using petroleum/ethyl acetate as an eluent to give themethoxy substituted arylethylyl trimethylsilanes.

(1) 2-(4-methoxyphenyl)-1-trimethylsilyl-ethyne

Purified by column chromatography on silica gel using petroleum ether asan eluent to give 2-(4-methoxyphenyl)-1-trimethylsilyl-ethyne (96%yield) as a light yellow oil.

(2) 2-(3,5-dimethoxyphenyl)-1-trimethylsilyl-ethyne

Purified by column chromatography on silica gel using petroleum ether asan eluent to give 2.2 g (94%) light yellow needles.

T_(GC)=5.39 (T_(init)=50° C.). ¹HNMR(CDCl₃, 300 Mz) δ ppm: 6.6(s, 2H,Ar—H), 6.43(s, 1H, Ar—H), 3.77(s, 6H, 2CH₃), 0.24(s,9H, SiMe₃).

C. Synthesis of Methoxy Substituted Arylacetylenes

To a solution of arylethynyltrimethylsilanes (30 mmol) in methanol (30ml) was added potassium fluoride (3.5 g, 60 mmol). The reaction mixturewas stirred at room temperature for 2 hours. After removal of methanol,the product was extracted with ether (100 ml×3) and purified bychromatography on silica gel using petroleum ether as eluent to affordpure products.

(1) p-Methoxyrthylnylbenzene

Pale yellow oil was obtained in 92% yield.

¹HNMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=8.98, Ar—H), 6.83(d, 2H,J=8.55, Ar—H), 3.80(s, 3H, Ch₃O), 3.00 (s, 1H—H).

(2) 3,5-Methoxyrthylnylbenzene

Pale yellow needle was obtained in 91 % yield.

¹HNMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=2.4, Ar—H), 6.83(d, 2H,J=2.3, Ar—H), 3.78(s, 6H, 2Ch₃O), 3.94 (s, 1H—H).

D. Synthesis of Methoxytolans

General Procedure

To a solution of methoxyethylnylbenzenes (20 mmol) and methoxysubstituted aryl iodide (22 mmol) in isopropylamine (120 ml) were addedPd(PPH₃)₂Cl₂ (0.2 mmol) and CuI (0.4 mmol). The reaction mixture wasstirred at ambient temperature for 6 hours under a slow stream ofnitrogen. The reaction mixture was filtered and the residues were washedwith ethyl acetate and the solvent evaporated from the combinedfiltrates. The crude product was purified by column chromatography onsilica gel using petroleum ether/ethyl acetate (9:1) as an eluent togive methoxytolans.

(1) 3,4′,5-Trimethoxyltolan

A pale yellow oil was obtained in 93% yield.

¹HNMR (CDCl₃) 300 Mz): δ ppm: 7.46(d, 2H, J=8.6, Ar—H), 6.88(d, 2H,J=8.8, A—H), d, 2H J=2.3, Ar—H), 6.44(t, 2H, J=2.3, Ar—H), 3.83(s, 3H,CH₃O), 3.80(s, 6H, 2CH₃O).

(2) 3,3′,5,5′-Tetramethoxytolan

A colorless needle crystal was obtained in 85% yield.

¹HNMR (CDCl₃) 300 Mz): δ ppm: 6.69(d, 4H, J=2.3, Ar—H), 6.46(d, 2H,J=2.3, Ar—H), 6.66(d, 2H J=2.3, Ar—H), 3.80(s, 12H, 4CH₃O).

(3) 4,4′-Dimethoxytolan

A colorless needle crystal was obtained in 91% yield.

¹HNMR (CDCl₃) 300 Mz): δ ppm: 7.46(d, 4H, J=8.7, Ar—H), 6.87(d, 2H,J=8.7, Ar—H), 3.82(s, 6H, 2CH₃O).

E. Synthesis of Hydroxytolans

General Procedure

In a dry 250 ml, 3-necked, round-bottomed flask was placed a solution ofmethoxytolans (10 mmol) in anhydrous methylene chloride under N₂. Thereaction mixture was cooled to below −20° C., and BBr₃ (20 mmol×thenumber of methoxy groups) by syringe. Then the reaction mixture waspermitted to warm up to room temperature and stirred for over 24 hours.The reaction mixture (a reddish clear solution) was then poured intoice-water and stirred. After sufficient stirring, an aqueous NaHCO₃solution was added to adjust the pH of the mixture to 7-8. Then themixture was extracted with ethyl acetate 3-4 times. The organic layerwas washed with brine and dried over MgSO₄. Solvent was removed underreduced pressure. The red brown color crude products was purified bycolumn chromatography on silica gel using petroleum/ethyl acetate (1:1)as en eluent to give hydroxytolans.

(1) 3,4′,5-Trihydroxytolan

A pale yellow solid was obtained in 82% yield.

¹HNMR (CDCl₃) 300 Mz): δ ppm: 9.89(s, 1H, OH), 9.45(s, 2 h, 2-OH,7.33(d, 2H, J=8.65, Ar—H), 6.78(d, 2H, J=8.63, Ar—H), 6.31(d, 2H, J=2.2,Ar—H), 6.23(d, 2H, J=2.2, Ar—H).

(2) 3,3,5,5′-Tetrahydroxytolan

A pale red solid was obtained in 92% yield.

¹HNMR (CDCl₃, 300 Mz): δ ppm: 9.49(s, 4H, 4-OH), 6.33(d, 4H, J=2.2,Ar—H), 6.25(t, 2H, J=2.2, Ar—H).

(3) 4,4′-Dihydroxytolan

A white solid was obtained in 93% yield.

¹HNMR (CDCl₃, 300 Mz): δ ppm: 9.82(s, 2H, 2-OH), 7.31(d, 4H, J=8.7,Ar—H), 6.77(d, 4H, J=8.7, Ar—H).

References

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EXAMPLE 2 Synthesis of 4-Hydroxy-4′-trifluoromethyltolan

FIG. 3 shows a synthetic scheme for the preparation ofhydroxy-trifluoromethyltolan. Synthetic details of the specific reactionsteps are described below. Most of the reactions were accomplished withhigh yields (over 90%). All products were purified by columnchromatography and characterized by GC and ¹HNMR spectrometry.

1. 1-Iodo-4-tetrahydropyranyloxybenzene 1

To a stirred solution of 4-iodophenol (11.0 g, 50 mmol) in CH₂Cl₂ (50ml) cooled with an ice bath, dihydropyran (5.0 g, 60 mmol) was addeddropwise over 10 min at 0-5° C. After the solution became clear,toluenesulfonic acid, TsOH, (10 mg) was added. The solution was stirredat 20° C. for 15 min. Then it was quenched by addition of NaHCO₃ (1 g)and 3 drops of water, and after stirring for 5 min at 20° C., thesolvent was removed in vacuo and the residue was purified by columnchromatography on silica gel with petroleum ether as eluent to give 14.0g (92%) of 1 as colorless crystal; mp 66° C.; δ_(H)(CDCl₃; 300 MHz):7.55(d, J=8.3, 2H, Ar—H), 6.83(d, J=8.4, 2H, Ar—H), 5.37(t, J=3.1, 1H,OCHO), 3.86(m, 1H, THP), 3.59(m, 1H, THP), 1.87˜1.58(m, 6H, THP).

2. 4-Tetrahydropyranyloxy-1-(trimethylsilylethynyl)benzene 2

To a degassed solution of compound 1 (9.12 g, 30 mmol) indiisopropylamine (180 ml) under nitrogen, Pd(PPh₃)₂Cl₂ (140 mg, 0.2mmol) and CuI (78 mg, 0.4 mmol) were added. Then trimethylsilylacetylene (3.3 g, 33 mmol) was added dropwise to this clear solution.The reaction mixture was stirred for 2 hours at room temperature. Thesalt formed during the reaction procedure was filtered off and washedwell with ethyl acetate. The filtrate was evaporated to dryness andhydrolyzed with concentrated hydrochloric acid (5 ml), water (25 ml) andcrushed ice (10 g), then extracted with ethyl acetate. The combinedorganic paste was washed with brine and dried with MgSO₄. The solventwas removed in vacuo and the residue was purified by columnchromatography (petroleum ether-ethyl acetate=9:1) to give a yellow oilof 2. Yield 7.9 g (96%); δ_(H)(CDCl₃; 300 MHz): 7.39(d, J=8.7, 2H, Ar—H,6.97(d, J=8.6, 2H, Ar—H), 5.41(t, J=3.1, 1H, OCHO), 3.84(m, 1H, THP),3.59(m, 1H, THP), 1.86˜1.61(m, 6H, THP), 0.23(s, 9H, 3CH3).

3. 4-Tetrahydropyranyloxyphenylacetylene 3

KF (9.3 g, 160 mmol) was added to a stirred solution of 2 (22.6 g, 80mmol) in MeOH (150 ml). The reaction mixture was stirred at roomtemperature for about 4 hours. After the reaction finshed (GC shows nostarting material remaining), the solvent was removed under reducedpressure on a rotary evaporator. The residue was purified by columnchromatography on silica gel (petroleum ether-ethyl acetate=9:1) to givea pale yellow crystals of 3. Yield 15.7 g(97%); mp 65° C., δ_(H)(CDCl₃;300 MHz): 7.42(d, J=8.7, 2H, Ar—H), 7.00(d, J=8.7, 2H, Ar—H), 5.43(t,J=3.2, 1H, OCHO), 3.87(m, 1H, THP, 3.60(m, 1H, THP), 2.99(s, 1H, C═C—H),1.96˜1.56(m, 6H, THP).

4. 4-Tetrahydropyranyloxy-4′-trifluormethyltolan 4

A solution of 3 (12.1 g, 60 mmol) and 4-bromobenzotriflouride (14.85 g,66 mmol) in diisopropylamine (250 ml) was heated to 30° C. undernitrogen, and the solution was degassed. Then Pd(PPh₃)₂Cl₂ (210 mg, 0.3mmol) and copper(I) iodide (114 mg, 0.6 mmol) were added to this clearsolution. The reaction mixture was stirred for 2 hours at 80° C., thencooled to room temperature. The salt formed during the reactionprocedure was filtered off and washed well with ethyl acetate. Thefiltrate was evaporated to dryness and hydrolyzed with concentratedhydrochloric acid (10 ml), water (100 ml) and crushed ice (50 g), thenextracted with ethyl acetate. The combined organic paste was washed withbrine and dried over MgSO₄. The solvent was removed in vacuo and theresidue was purified by column chromatography (petroleum ether-ethylacetate=9:1) to give a pale yellow crystals of 4. Yield 16.6 g (80%); mp112˜113° C.; δ_(H)CDCl₃; 300 MHz): 7.59(s, 4H, Ar—H), 7.48(d, J=8.7, 2H,Ar—H), 7.04(d, J=8.7, 2H, Ar—H), 5.46(t, J=3.1, 1H, OCHO), 3.89(m, 1H,THP), 3.62(m, 1H, THP), 1.86˜1.62(m, 6H, THP).

5. 5-Hydroxy-4′-trifluoromethyltolan 5

Compound 4 (13.84 g, 40 mmol), CH₂Cl₂ (75 ml) and MeOH (125 ml) wereplaced in a 250 ml round bottomed flask, then TsOH (0.4 g, 0.4 mmol) wasadded. The reaction mixture was stirred at 30° C. for 1 hour. When thereaction was finished (TLC shows no starting material remaining), thesolvent was removed by rotary evaporation and the residue was dissolvedin EtOAc and filtered through silica gel. The solvent was removed andthe solid was recrystallized from solvents of ethyl acetate and hexane(1:5) to give a pale yellow crystal 9.5 g (90%), mp 131-132° C.,δ_(H)(CDCl₃; 300 MHz): 7.59(s, 4H, Ar—H), 7.44(d, J=8.7, 2H, Ar—H),6.82(d, J=8.7, 2H, Ar—H), 5.16(s, 1H, OH).

References

1 Shen, D., Diele, S., Pelzl, G., Wirth, I. and Tschierske, C., J.Matter. Chem., 1999, 9, 661.

2 Praefcke, K., Kohne, B. and Singer, D., Angew. Chem. Int. Ed. Engl.,1990, 29, 177.

3 Bouchta, A., Nguyen, H. t., Achard, M. F., et al., Liq. Crystals,1992, 12, 575.

4 Hsieh, C. J. and Hsiue, G. H., Liq. Crystals, 1994, 16, 469.

EXAMPLE 3 Inhibiting Formation of Infectious HSV-1 Particles byTreatment With a Di-Hydroxylated Tolan

Cultures of African green monkey kidney cells (Vero) cells, obtainedfrom the American Type Culture Collection, Rockville, Md., were grown toconfluence in Medium 199 supplemented with 5% fetal bovine serum, 0.075%NaHCO₃, and 50 μg/ml gentamycin sulfate in 25 cm² tissue culture flasks.Cells were infected with HSV-1 at a multiplicity of infection (moi) ofone and incubated at room temperature for one hour to allow for virusattachment to and penetration of the cell. Under these conditions,approximately half of the cells are infected with virus. Thereafter, thecultures were rinsed three time with media and incubated in mediumcontaining 50 μM or 75 μM 4,4′-dihydroxytolan prepared in 0.2%dimethyl-sulfoxide (DMSO). Controls were treated identically, but wereincubated without the dihyroxylated tolan. For purposes of comparison,HSV cell were also inoculated in the presence of 52.5 μM4,4′-dihydroxystilbene. Due to the higher cell toxicity of the stilbene,cells were not incubated in the presence of 75 μM dihydroxystilbene.

Upon addition of the medium to the cultures and at 24 hours time periodsthereafter, i.e., 0 hours, 24 hours, 48 hours, and 72 hours afteraddition of the drug, cells and medium were frozen at −70° C. Sampleswere then thawed, sonicated and titrated on Vero cells to determine thenumber of plaque forming units (pfu's) of virus produced by eachculture.

As shown in FIG. 4, the number of pfu's produced in the control culturesinfected with an moi of 1 reaches peak production at approximately 24hours after infection. At this time, the system is exhausted, i.e.,active virus has infected and destroyed not only those cells infectedduring the initial one hour of incubation but also those cells whichbecame infected with virus released by the initially-infected cells. Thelack of increase observed in the control cultures at 72 hours treatmentindicates that the virus production has peaked, due to the lack ofviable cells in which to reproduce.

As shown in FIG. 4, treatment of cells with 75 μM 4,4′-dihydroxytolaninhibited formation of infectious virus particles in HSV-1 infectedcells by more than 99% at 24 hours. By 72 hours, infectious HSVparticles were virtually undetectable in cultures continuously incubatedin the presence of 75 μM 4,4′-dihydroxytolan, Treatment with 50 μM4,4′-dihydroxytolan reduced new virus production by only 95%. Incontrast, treatment of the HSV infected cells with 50 μM4,4′-dihydroxystilbene had no effect on virus production. (data notshown) These results also demonstrate that inhibition of virusreplication by the dihydroxylated tolan is dose dependent.

EXAMPLE 4 Inhibiting Formation of Infectious HSV-1 Particles byContacting Cells with a Hydroxylated Tolan Prior to or During an EarlyStage in Replication

Vero cell cultures were infected with HSV-1 as described above inExample 3 except that the cells were infected with virus at an moi of10. Under these conditions nearly all of the cells are infected withvirus during the initial one hour incubation period. Following removalof unattached virus, the virally-infected cultures were incubated incontrol medium lacking a hydroxylated tolan or medium to which 105 μM4,4′-dihydroxytolan had been added at 1, 3, 6, or 9 hours after removalof the unattached virus. At 24 hours after infection, the number ofpfu's present in the cells and medium of untreated and hydroxylatedtolan-treated cultures was determined.

The results presented in FIG. 5 demonstrate that the hydroxylated tolanis most effective when administered to virally-infected cells during theearly stages of viral replication. In cultures treated with 105 μM4,4′-dihydroxytolan at one hour after infection, production of virus wasreduced by more than 99%. In cultures treated with 105 μM4,4′-dihydroxytolan at 3 or 6 hours after infection, the production ofvirus was inhibited by approximately 90%. When the hydroxylated tolanwas added 9 hours after infection, formation of infectious virusparticles was not inhibited.

To determine whether hydroxylated tolans block formation of infectiousherpes virus particles by directly inactivating the virus, a standardinoculum of HSV-1 was mixed with 105 μM 4,4′-dihydroxytolan in medium,with 0.2% DMSO in medium, or with media alone and placed at roomtemperature. The number of residual pfu's present at 1, 10, 30, and 60minutes after addition of each respective solution to the virus wasdetermined by plaque assay. The results demonstrated that thehydroxylated tolan did not directly inactivate HSV (data not shown).

Studies also demonstrated that the hydroxylated tolan did not preventattachment of HSV-1 to cells. (data not shown)

EXAMPLE 5 Inhibiting HSV Replication

Vero cells were grown to confluence and infected with HSV-1 at an moi of1 and then incubated in media lacking a hydroxylated tolan (controlcultures) or in media containing 105 μM 4,4′-dihydroxytolan. One set ofinfected cells was maintained in the dihydroxylatedtolan for a period of72 hours. In another set of infected cells the dihydroxytolan-containingmedia was replaced with media lacking a hydroxylated tolan at 24 hours.In another set of cells the tolan-containing media was replaced withmedia lacking the hydroxylated tolan at 48 hours after infection. Thenumber of infectious HSV particles produced by each set of infectedcells was determined by plaque assay.

The results shown in FIG. 6 demonstrate that the inhibitory effect of105 μM 4,4′-dihydroxytolan on HSV replication in virus infected cells isreversible. Accordingly, continuous treatment of HSV-infected cells withthe hydroxylated tolan maintains the virus in a non-infectious state.Discontinuing treatment with the hydroxylated tolan allows replicationof the virus to proceed in what appears to be a normal fashion. Theresults presented in FIG. 6 also suggest that HSV replication in thehydroxylated tolan treated cells was blocked at an early phase, i.e.,replication of HSV had not progressed past the stage where cells are sodamaged that they are unable to support replication of this herpesvirus.

The results presented in FIG. 6 also indicate that exposure of mammaliancells to 105 μM 4,4′-dihydroxytolan for a prolonged period of time doesnot kill the cells. Cell viability studies confirmed that treatment ofuninfected Vero cells with 105 μM 4,4′-dihydroxytolan for 24 hours wasnot toxic.

EXAMPLE 6 Characterizing Viral Proteins Produced in the Presence of aPolyhydroxylated Tolan

ICP-4 and ICP-27 are immediate-early regulatory proteins of HSV-1 thatare required for efficient replication of this virus. To determinewhether ICP-4 or ICP-27 production is altered by treatment with ahydroxylated tolan, separate cultures of Vero cells were infected withHSV-1 at an moi of 1 and incubated in control medium or mediumcontaining 105 μM 4,4′-dihydroxytolan for 24 hours. Infected cells werescraped from the flask, collected by centrifugation, and resuspended incold tris-buffered saline, pelleted by centrifugation, and the cellpellet frozen at −70° C. Proteins were extracted from the thawedpellets, separated by 6-15% SDS-PAGE, and assayed on a Western blot byreacting with mouse monoclonal antibody to ICP-4 or ICP-27 from GoodwinInstitute for Cancer Research Inc., FL.

As shown in FIGS. 7 and 8, treatment of HSV-infected cells with thedihydroxytolan significantly reduced synthesis of ICP-4 and ICP-27, twomajor regulatory protein. These results confirm that treatment with ahydroxylated-tolan inhibits synthesis of herpes viruses at an earlyphase in the replicative scheme. These results also indicate thatcultured cells treated with a hydroxylated tolan are a useful modelsystem for characterizing the herpes virus gene products that are madeduring the immediate early phase and early phase of HSV replication.

EXAMPLE 8 Cell Toxicity of Tolans

Cell toxicity of the tolans 4,4′-dihydroxytolan (“Tolan-5”),3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (“Tolan-11”) was determined using anMTT assay. In this assay, cells are exposed to MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which istaken into the cells and reduced by mitochondrial dehydrogenase to apurple formazan, a large molecule which is unable to pass through intactcell membranes, and therefore accumulates in healthy cells. The abilityof cells to reduce MTT is an indication of mitochondrial integrity andactivity, which may be interpreted as a measure of viability.Solubilization of the cells results in the liberation of the productwhich can readily be detected spectrophotometrically.

Various concentrations of Tolan-5, Tolan-10 and Tolan-11 were added to96 well tissue culture plates that had been seeded with Vero cells. Theplates were then incubated at 37° C. in 5% CO₂/95% air for 24, 48 and 72hours. At the end of each time point, 50 μl of MTT (1 mg/ml) was addedto the tissue culture plates which were then incubated for an additionalfour hours. At the end of that time, dimethyl sulfoxide (DMSO) was addedto dissolve the cells and formazan, and the plates were readspectrophotometrically at 540 nm. From this data a CD₅₀ or theconcentration of the respective tolan required to render 50% of thecells non-viable was calculated. Presented in Table I below are thetoxicity results for Tolan-5, Tolan-10, and Tolan-11 measured at 24, 48and 72 hours of treatment.

TABLE 1 CD₅₀ for hydroxylated Tolans of Examples 1 and 2: Toxicity wasmeasured using a MTT colorimetric assay at 48 hours in Vero cells, andreported as the cytotoxic dose (CD₅₀ in μM, based on a 50% reduction incell viability. CD₅₀ in μM Time in hours Name 24 48 72 Tolan-5 — 10684.0 Tolan-10 >200 >200 >200 Tolan-11 72.1 48.2 47.1

As shown in Table 1, Tolan-10 showed very little toxicity. The estimatedCD₅₀ for this trihydoxytolan is >200 μm. The hydroxytrfluoromethyltolan,Tolan-11, was more toxic than the other hydroxylated tolans and is,thus, less preferred.

EXAMPLE 9 Inhibition of HSV-1 Replication In Vitro

Tissue culture cells were infected with HSV-1 and incubated in solutionscontaining Tolan-5, Tolan-10 and Tolan-11. The concentrations usedranged from one-half to three-quarters of the calculated CD₅₀ for therespective tolan at 48 hours of treatment.

Specifically, Vero cells were infected with HSV-1 at a multiplicity ofinfection of one for one hour. At the end of that time, the cultureswere rinsed with media and fresh media containing various concentrationof the respective tolan were added. Infected control cultures wereincubated in media lacking hydroxylated tolan. At 1, 24, 48, and 72hours after infection, samples were frozen at −70° C. until assayed fornew virus production. Virus production was quantified by the plaqueassay for each time point of infection. The results, which are expressedas plaque forming units per milliliter (pfu/ml).

As shown in FIGS. 9A-C, Tolan-5, Tolan-10, and Tolan-11 all inhibitedHSV-1 production, but to varying degrees. Tolan-5 (FIG. 9A) was moreinhibitory to the virus at 75 μm that at 50 μm. Tolan-10 (FIG. 9B) wasequally effective against the virus at all concentrations, which rangedfrom a high of 350 μm to a low of 175 μm. Tolan-11 had little to noeffect at concentration of less than 48 μm.

When inhibition occurred, it was seen with all tolans at the earliesttime point assayed, which was 24 hours after cell infection. Oncesuppressed, the virus did not recover over the 72 hour time period thecells were tested for virus production.

EXAMPLE 10 Inhibition of HSV In Vivo

Female SKH-1 hairless mice 4-5 weeks old were lightly anesthetized and ascratch approximately 2-3 mm long and 0.25 mm deep was made on thelateral dorsal aspect of the neck. The scratch was infected with 10⁶plaque forming units of HSV-1. One hour later, the infected scratch areawas treated with a 5% or 10% solution of Tolan-10 in DMSO. Thistreatment was repeated three times a day for five days. There were atleast six mice in each treatment group. Untreated mice and mice treatedwith DMSO only were used as controls.

Beginning on day 1 and continuing for 10-11 days, the scratch site wasexamined daily for evidence of an HSV infection and scored according tothe following schedule:

0=no visible change on skin by scratch

1+=papules around scratch

2+=papules around scratch ulcerate with scab formation

3+=ulcerative zosterform spread along peripheral nerves to flank ofanimal

4+=ulcerative lesions open on flank

5+=death/sacrifice

The data was grouped according to treatment and statistically analyzedfor significance.

As shown in FIGS. 10 and 11, Tolan-10 at concentrations of 5 and 10%significantly reduced the severity of HSV lesions when compared tountreated controls or animals that were treated with DMSO only. Thisdifference was evident as early as three days after infection. Althoughnot significantly different, 10% Tolan-10 appeared to be slightly moreeffective than 5% Tolan-10, particularly on days 6-10. At day 11,however, there was no difference between the two treatment groups.

Survival data is shown in FIG. 11. While 50% of the animals in the DMSOcontrol and no treatment control groups died within 11 days afterinfection, none of the animals treated with 5% or 10% tolan perishedduring this time period.

EXAMPLE 11 Inhibiting Growth of Neisseria gonorrhea by Treatment withTolan-10

Various concentrations of the trihydroxytolan Tolan-10 were incorporatedinto chocolate agar plates that were prepared using standard procedures.The trihydroxylated tolan was first dissolved in DMSO and medium andthen added to liquid chocolate agar, which was then poured into petriplates and allowed to solidify. The highest concentration of DMSO in theagar was 0.5%. Control plates containing chocolate agar and DMSO at afinal concentration of 0.5% were also prepared.

Neisseria gonorrhea CDC 98 was obtained from Difco Laboratories. Theauthenticity of the bacterium was confirmed utilizing standardmicrobiological techniques of identification. Cultures of the bacterialisolate were inoculated onto fresh plates of solidified chocolate agarand then 24 hours later, a suspension was made from isolated colonies.

10 μl aliquots of the suspension were spread evenly across the surfaceof solidified control chocolate agar lacking tolan and the surface ofsolidified chocolate agar containing Tolan-10 at final concentrationsranging from 1 to 125 μg/ml. Thereafter, the agar plates were incubatedat 37° C. with or without 5% CO₂. All plates were visually examined forgrowth of the bacterium 24 hours later to determine the concentration ofthe tri-hydroxylated tolan that inhibits growth by 50% (MIC₅₀) as wellas the concentration which inhibits any visible growth (MIC₁₀₀).

The effect of the same concentrations of Tolan-10 on the growth ofEscherichia coli, Staphylococcus aureus, Streptococcus pyogenes,Pseudomonas aeruginosa, Neisseria meningiditis and Candida albicans wasalso determined. As shown in Table II below, Tolan 10 selectivelyinhibited Neisseria gonorrhoeae. The IC₅₀ value of the tri-hydroxylatedtolan for this bacterium at 24 hours of treatment was 50 μg/ml and theIC₁₀₀ value was 100 μg/ml. In contrast, Tolan 10, at the highestconcentration tested, had no effect on the other microorganisms listed.

TABLE 2 IC's of Tolan-10 against bacteria and C. albicans MicroorganismIC₅₀ (mg/L) IC₁₀₀ (mg/L) N. gonorrhoeae 50 100 N. meningitides >125 >125E. coli >125 >125 S. aureus >125 >125 S. pyogenes >125 >125 P.aeruginosa >125 >125 C. albicans >125 >125

What is claimed is:
 1. A method of treating a subject having orsuspected of having an infection induced by a herpes virus selected fromthe group consisting of HSV-1, HSV-2, CMV and VZV, said methodcomprising administering a pharmaceutical composition comprising atherapeutically effective amount of a hydroxylated tolan to the subject,wherein the hydroxylated tolan is a di-hydroxylated tolan or atri-hydroxylated tolan.
 2. The method of claim 1 wherein thepharmaceutical composition further comprises a topical carrier andadministration is by topical administration to the skin.
 3. The methodof claim 1 wherein administration is to the eye.
 4. The method of claim1 wherein administration is to the oral cavity or lips.
 5. The method ofclaim 1 wherein administration is by vaginal insertion or analinsertion.
 6. The method of claim 2 wherein the herpes virus infectionis caused by HSV-1 or HSV-2.
 7. The method of claim 1 wherein thepharmaceutical composition is administered to an infected site duringthe prodromal stage of infection.
 8. The method of claim 1 wherein thepharmaceutical is applied at or proximate a known site of infection or asite which is suspected of being infected.
 9. A topical composition forreducing the symptoms of a herpes virus infection, said herpes virusbeing selected from the group consisting HSV-1, HSV-2, VZV and CMV, saidtopical composition comprising: (a) a therapeutically effective amountof a hydroxylated tolan; and (b) a pharmaceutically acceptable carrier,wherein the hydroxylated tolan is 4,4′-dihydroxytolan,3,4′,5-trihydroxytolan, or a combination of 4,4′-dihydroxytolan and3,4′,5-trihydroxytolan.
 10. The topical composition of claim 9 whereinsaid topical composition is formulated for administration to the skin,reproductive tract, oral cavity or eye of a subject in need of the same.